Project 5 ? Double-Strand Break Repair and Pathway Choice PROJECT SUMMARY/ABSTRACT DNA double strand breaks (DSBs) are the most deleterious form of DNA damage, and failure to repair DSBs leads to cell death or chromosomal instability that is a hallmark of cancer. DSB detection, signaling and initiation of one of three distinct DSB repair pathways are critical processes for the cell's fate and genomic integrity. Project 5 integrates into SBDR-4 by focusing on dynamic complexes acting in DSB Repair (DSBR) initiation and signaling, non-homologous end joining (NHEJ) progression, and pathway choice between homologous recombination repair (HRR) and NHEJ. DSBR is directly relevant to cancer biology and interventions. The impact of selecting between HRR, which is error-free, and NHEJ, which can be error-prone, depends upon cell cycle status and the nature of the damage. Current chemotherapy and radiotherapy create DSBs, and PARP inhibitors specifically kill cancer cells that are DSBR deficient through synthetic lethality. Furthermore, the specific targeting of cancer cell susceptibilities by selectively blocking specific DSBR responses provides a promising approach to kill cancer cells with less damage to normal cells. Yet, without a structural and mechanistic knowledge of DSB repair, our ability to interpret mutations arising from the clinic and to provide knowledge for improved design of future therapies is limited. Project 5 will characterize macromolecular interfaces, assemblies, conformations, and key phosphorylation and ubiquitination events underlying DSBR initiation, regulation and pathway choice. In Aim 1, MRN-DNA interactions will be defined, and we will determine structures for MRN, CtIP and ATM activation and regulation. We will define their key roles in initiating HRR, to identify mutations and inhibitors to dissect multi-functionality and to uncover synthetic lethality. In Aim 2, we will define NHEJ interfaces, focusing on the PNKP-XRCC4-LigIV complexes and the nuclease Artemis to define their critical roles in NHEJ. In Aim 3 we will focus on the heart of pathway choice by determining how DNA-PK and MRN compete for DNA in the context of chromatin, and via the post- translational modifications of phosphorylation and ubiquitination that regulate NHEJ and HRR initiation and signaling. Project 5 requires extensive use of the EMB core for constructs and protein complex expression, and of the SCB Core for structural experiments. Project 5 will exchange mutants, constructs, inhibitors and other results with Projects 2-4 and collaborate to examine impacts on multiple DNA repair pathways with Project 1. The anticipated outcomes of the proposed experiments include an actionable mechanistic knowledge to inform the Cancer Genome Atlas and system level studies of knockouts. This knowledge will help to achieve NCI missions by bridging the gap from sequences and mutations to phenotype prediction by defining biologically validated atomic complexes, conformations, and mechanisms for synthetic lethality.